Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme.
The bovine pancreatic (bp-) DNase I gene has been cloned from bp-cDNA and expressed in E. coli. A polynucleotide sequence of 1295 base pairs was deduced from clones of the cDNA. The sequence showed an open reading frame which can be translated as a 282-amino acid polypeptide, including a hydrophobic signal peptide and the polypeptide of bp-DNase I. An expression plasmid was constructed by inserting into the vector pET-15b, a cDNA fragment coding for bp-DNase I ligated with a hexanucleotide coding for Met-Ala at the 5'-end. The plasmid was transformed into E. coli strain DH5alpha and the active recombinant bovine (rb-) DNase I was produced after induction of protein synthesis. From the induced culture medium, rb-DNase I was purified by chromatography on a Mono Q column. The purified rb-DNase I showed a molecular mass of 29 kDa and had the same specific activity as bp-DNase I. The NH2-terminus of rb-DNase I was Ala, not Met, and at position 19, corresponding to the carbohydrate attachment site of bp-DNase I, Asn was not glycosylated.
Chen
C Y
CY
Institute of Biochemistry, College of Medicine, National Taiwan University, No.1, Sec. 1, Jen-Ai Road, Taipei, Taiwan, People's Republic of China.
Lu
S C
SC
Liao
T H
TH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000138:chemical synthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I